Angiogenesis promoted by caged growth factors

ABSTRACT

The present disclosure relates to controlling the release of growth factors for the promotion of angiogenesis. The growth factors or a polymer matrix are modified by photoactive compounds, such that the growth factors are not released into an active form until they are irradiated with light. The disclosure also relates to tissue engineering scaffolds comprising one or more polymers and at least two growth factors.

TECHNICAL FIELD

This disclosure relates generally to the fields of vascularization andtissue engineering, including scaffolds.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Blood vessels are assembled by two processes known as vasculogenesis andangiogenesis. In vasculogenesis, a primitive vascular network isestablished during embryonic development from endothelial cellprecursors called angioblasts. Angiogenesis involves preexisting vesselssending out capillary buds or sprouts to produce new vessels.Angiogenesis is an important process critical to chronic inflammationand fibrosis, to tumor cell growth, and to the formation of collateralcirculation. Angiogenesis is involved in the normal process of tissuerepair.

Tissue engineering involves the use of living cells to developbiological substitutes for tissue replacement. However, in order fortissue engineering to be practical, scaffolds must be developed thatallow for tissue growth that approximates natural tissue growth.Vascularization is necessary to enable tissue engineering to be used inapplications that require structures greater than 0.5 mm thick. Thecontrolled growth of vascular networks requires the timed release ofmultiple growth factors throughout the maturation process. In vivo, therelative timing, quantity, and location of growth factor release isregulated as part of a complex biological system.

SUMMARY

The present disclosure relates to controlling the release of growthfactors for the promotion of angiogenesis. In one aspect, the disclosureprovides a method for promoting angiogenesis at a target site in amammalian subject comprising administering to a mammalian subject aformulation comprising at least two growth factors, wherein a firstgrowth factor is released in a bioactive state in response to a firstspectral sensitivity range and a second growth factor is released in abioactive state in response to a second spectral sensitivity range;irradiating the target site with a first wavelength of light in thefirst spectral sensitivity range to release the first growth factor; andirradiating the target site with a second wavelength of light in thesecond spectral sensitivity range to release the second growth factor;wherein the sequential administration of the first growth factor and thesecond growth factor promotes angiogenesis at the target site.

In one embodiment, one or more of the at least two growth factors arecaged. In one embodiment, one or more caged growth factors are notreleased in a bioactive state until radiated.

In one embodiment, the formulation further comprises one or morepolymers. In illustrative embodiments, the one or more polymers areselected from the group consisting of poly(lactic acid) polymers,poly(glycolic acid)polymers, poly(lactide-co-glycolides) (PLGA),poly(urethanes), poly(siloxanes) or silicones, poly(ethylene),poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate),poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinylalcohol) (PVA), poly(acrylic acid), poly(vinyl acetate), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polylactic acid (PLA), polyglycolic acids (PGA), nylons,polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol) (EVOH),polycaprolactone, poly(vinyl acetate), polyvinylhydroxide, poly(ethyleneoxide) (PEO), and polyorthoesters or a co-polymer formed from at leasttwo members of the group.

In an illustrative embodiment, the one or more polymers comprise aphotoactive group that cleaves the polymer in response to light therebyreleasing a growth factor entrained therein. In one embodiment, the oneor more polymers comprise a group sensitive to degradation by aphotoacid.

In one embodiment, the first and second spectral sensitivity ranges arenot substantially overlapping. In one embodiment, an amplitude of theirradiating of the target site controls a rate of release of the firstgrowth factor, the second growth factor, or both. In one embodiment, theirradiating comprises illumination with a laser, a mercury bulb, or anelectromagnetic radiation source in combination with a filter.

In one embodiment, the at least two growth factors are independentlyselected from the group consisting of: transforming growth factor-alpha(TGF-α), transforming growth factor-beta (TGF-β), platelet-derivedgrowth factor (PDGF), fibroblast growth factor (FGF), nerve growthfactor (NGF), brain derived neurotrophic factor, cartilage derivedfactor, bone growth factor (BGF), basic fibroblast growth factor,insulin-like growth factor (IGF), vascular endothelial growth factor(VEGF), granulocyte colony stimulating factor (G-CSF), hepatocyte growthfactor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF),keratinocyte growth factor (KGF), and skeletal growth factor. In anillustrative embodiment, the first growth factor is VEGF and the secondgrowth factor is PDGF.

In one aspect, the disclosure provides a tissue engineering scaffoldcomprising: one or more polymers and at least two growth factors,wherein a first polymer is configured to release a first growth factorin a bioactive state in response to a first spectral sensitivity rangeand a second polymer is configured to release a second growth factor ina bioactive state in response to a second spectral sensitivity range.

In one embodiment, the scaffold further comprises collagen. In oneembodiment, the scaffold further comprises elastin.

In one aspect, the disclosure provides a method of controlling therelease of two or more growth factors at a target site, comprising:delivering a tissue engineering scaffold to a target site; releasing afirst growth factor at the target site by radiation of the target sitewith a first wavelength of light in the first spectral sensitivityrange; and releasing a second growth factor at the target site byradiation of the target site with a second wavelength of light in thesecond spectral sensitivity range. In one embodiment, an amplitude ofthe radiation of the target site controls a rate of release of the firstgrowth factor, the second growth factor, or both.

In one aspect, the disclosure provides a method for tissue engineeringin a mammal, comprising: applying to a tissue progenitor site of amammal an effective amount of a scaffold comprising one or more polymersand at least two growth factors, wherein a first polymer is configuredto release a first growth factor in a bioactive state in response to afirst spectral sensitivity range and a second polymer is configured torelease a second growth factor in a bioactive state in response to asecond spectral sensitivity range.

In one embodiment, the scaffold further comprises a population of cells.In one embodiment, the scaffold comprises a population of vascularendothelial cells or cell precursors and wherein application of thescaffold to the tissue progenitor site stimulates vascularization in thesite.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the following drawings and thedetailed description.

DETAILED DESCRIPTION

The illustrative embodiments described in the detailed description andclaims are not meant to be limiting. Other embodiments may be utilized,and other changes may be made, without departing from the spirit orscope of the subject matter presented here.

The present technology is described herein using several definitions, asset forth throughout the specification. As used herein, unless otherwisestated, the singular forms “a,” “an,” and “the” include pluralreference. Thus, for example, a reference to “a polymer” includes a oneor more polymers.

As used herein, the “administration” of an agent or drug to a subjectincludes any route of introducing or delivering to a subject a compoundto perform its intended function. Administration can be carried out byany suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “bioactive state” refers to the form of acompound or agent which is capable of inducing a desired biological orpharmacological effect, which may include but is not limited toangiogenesis. The effect may be local, such as providing for a localneovascular effect in a target site for tissue repair.

As used herein, the term “caged growth factor” refers to a growth factorwhose biological activity is controlled by light, typically by thephotolytic conversion from an inactive to an active form.

As used herein, the term “biocompatible polymer” refers to a syntheticor natural material that is compatible (i.e., non-toxic) to biologicalsystems. A “biodegradable, biocompatible polymer” refers to abiocompatible polymer that will degrade (i.e., break down) when exposedto, or placed in, a biological system. The rate of degradation may befast (e.g., degradation may take place in minutes) or slow (e.g.,degradation may take place over hours, days, weeks or months), or thepolymer may degrade in response to a particular stimulus, e.g.,irradiation with light. In some embodiments, the rate of degradation maybe controlled by the type of polymer used and/or the amplitude of lightapplied to the polymer.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic effect, e.g., an amount of agrowth factor which results in angiogenesis in a target tissue. Theamount of a composition administered to the subject will depend oncharacteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic compounds.

As used herein, the term “irradiation” or “irradiating” is usedexpansively to encompass bombardment of the target site with photons,e.g. visible light, ultraviolet (“UV”) radiation, combinations thereof,and the like, in order to effect conversion of a photosensitivematerial.

As used herein, the term “polymer” refers to a macromolecule made ofrepeating (monomer) units or multimers.

As used herein, the term “spectral sensitivity range” refers to thespectral regions in which a photosensitive material is structurallytransformed or changed in response to radiation.

As used herein, the term “tissue scaffold” refers to any compositionformed into a porous matrix into which tissue can grow in threedimensions.

As used herein, the terms “scaffolding polymers” or “scaffoldingmaterials” refer to the materials used to make tissue scaffolds. Theterms refer to both monomeric units of the materials and the polymersmade therefrom. Scaffolding polymers may be biodegradable or nonbiodegradable.

Overview

Vascularization is a complex process that is regulated by multiplegrowth factors, which are each released at different times during thematuration of a blood vessel. These timings will not be identical forblood vessels of different sizes, and at different locations in a tissueengineering construct. The compositions and methods described hereinallow for the release of multiple growth factors to be regulated in timeand space.

The molecular mechanisms controlling the formation of mature vasculatureinvolve several sequential factors, each playing a distinct role.Controlled delivery of at least two growth factors (e.g., VEGF and PDGF)initiates formation of a blood vessels and induces their maturation.Thus, therapeutic angiogenesis benefits from the actions at least twotypes of molecules: initiation of blood vessels, as provided by VEGF orother factors (e.g., angiopoietin-2), and the maturation of bloodvessels by PGDF or other factors (e.g., angiopoietin-1). In oneembodiment, separate controlled release formulations of VEGF and PDGFare created, such that VEGF is released before PDGF.

Thus, in one aspect, the present disclosure relates to controlling therelease of growth factors for the promotion of angiogenesis. Inillustrative embodiments, either the growth factors or the polymermatrix are modified by photoactive compounds, such that the growthfactors are not released until they are irradiated with light. Thisapproach allows for control of (1) the timing, (2) the location, and (3)the amount of growth factor release. More than one growth factor can bereleased by irradiation, e.g., each by a separate wavelength, allowingfor the release of multiple chemicals to proceed with multiple timingsat different locations. Spatial control of growth factor release can beimportant, as different regions in a tissue engineering construct maymature at different rates due to differences in mass transport. Existingmethods that rely on standard controlled release of drugs cannotaccommodate this variability.

In some embodiments, growth factors may be released in a desired spaceby irradiating the region with light. The timing of the release is thuscontrolled by determining when the irradiation starts, the rate ofrelease is controlled by modulating the amplitude of the irradiation,and the location of the release is determined by focusing and/or maskingthe incoming light. For instance, each growth factor may be releasedusing different photoactive molecules, having non-overlappingabsorbances. As a result, the disclosure enables control over therelease kinetics of multiple growth factors at different rates inoverlapping regions of space.

In one embodiment, the growth factors themselves can be “caged”, orderivitized with a molecule such that they are not released in abioactive state until irradiated. In another embodiment, the growthfactors are entrained within a photosensitive polymer, which may bedegraded in response to exposure to light, thereby releasing the growthfactor. Thus, in the first embodiment, the growth factor itself isactivated in response to light; in the second embodiment, a capsule orpolymer containing the growth factor is exposed to light, therebyreleasing an active growth factor.

In one embodiment, two growth factors (e.g., VEGF and PDGF) areentrained in a photodegradable polymer by derivatizing two sets of highmolecular weight biocompatible polymer (e.g., PLA) with two differentphotoactive groups; encapsulating the growth factors in each of the twopolymers to form two separate powders; and fusing the powders togetherto form a porous tissue engineering scaffold.

In one embodiment, three growth factors are entrained in aphotodegradable polymer according to the following method: derivatizingthree sets of high molecular weight biocompatible polymer (e.g., PLA)with three different photoactive groups of orthogonal photoactivity;encapsulating three separate growth factors in each of the threepolymers, in order to form three separate powders; and fusing thepowders together to form a porous tissue engineering scaffold.

These powders can be optionally supplemented by a conventional molecularweight polymer of the same chemical composition, to dilute the totalamount of encapsulated material. After cell seeding, immature vascularnetworks will be physically surrounded by all three growth factors, butnone will be released until stimulated with the appropriate wavelengthof light.

Caged Growth Factors

One embodiment is directed to a growth factor bonded to a photoreactivemoiety. The resulting conjugate can be selectively cleaved to releasethe active growth factor. Cleavage, as referred to herein, is byphotocleavage or a cleavage event triggered by the application ofradiation to the conjugate.

In some embodiments, the photoreactive moiety is a chemical moietycapable of interacting directly or indirectly with a therapeutic agentwhich can be cleaved with electromagnetic radiation. The photoreactivemoeity may be positioned on the therapeutic agent (e.g., growth factor)so as to interfere with or prevent the normal biological activity of theagent. For instance, in the case of a polypeptide, the photoreactivemoiety may be positioned near the active site or substrate bindingdomain of the protein, which blocks the activity of the polypeptide.Suitable photoreactive moieties are generally selected for absorption oflight that is deliverable from common radiation sources (e.g. UV lightranging from 240-370 nm). Examples of photoreactive moieties which arephotoresponsive to such wavelengths include, but are not limited to,acridines, nitroaromatics and arylsulfonamides.

The efficiency and wavelength at which the photoreactive moiety becomesphotoactivated and thus releases or “uncages” the therapeutic agent willvary depending on the particular functional group(s) attached to thephotoreactive moiety. For example, when using nitroaromatics, such asderivatives of o-nitrobenzylic compounds, the absorption wavelength canbe significantly lengthened by addition of methoxy groups. In oneembodiment, nitrobenzyl (NB) and nitrophenylethyl (NPE) is modified byaddition of two methoxy residues into 4,5-dimethoxy-2-nitrobenzyl (DMNB)and 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE), respectively, therebyincreasing the absorption wavelength range to 340-360 nm (λ_(max)=355nm).

In one embodiment, the photoreactive moiety is a 2-nitrobenzylderivative. In their ground state, 2-nitrobenzyl-based agents andconjugates have an intramolecular hydrogen bond between benzylichydrogen and the ortho nitro group. Upon illumination with wavelengthsof greater than 300 nm, these chemical compounds transition to anexcited state. Proton transfer reaction from benzylic carbon to theoxygen in nitro group takes place which is followed by electronrearrangement. This reaction results in the formation of a transientspecies called an aci-nitro ion which is in a rapid equilibrium with acyclic form. In the cyclic intermediate, electron rearrangement andoxygen transfer from nitrogen to benzylic carbon takes place resultingin the formation of 2-nitroso derivatives and release of a substratewhich is a good leaving group.

In one embodiment, the photocleavable moiety is 3,5-dimethoxybenzyl or2-nitrobenzenesulfenyl. These nitrobenzyl groups all contain a benzyliccarbon-hydrogen bond ortho to a nitro group, which is necessary fortheir photolability.

In one embodiment, the photoreactive moiety is a photocleavable biotin(PCB). A wide variety of biotinyl moieties can be used to form a PCBmolecule. Biotin is comprised of a ring linked to an alkyl chainterminated by a carboxyl group. Numerous modifications can be made tothe biotin moiety which involve changes in the ring, spacer arm andterminating group, all of which still exhibit a high affinity forstreptavidin, avidin and their derivatives. Examples of photocleavablebiotins are known in the art. Unlike conventional biotins,photocleavable biotins enable one to release or elute the boundsubstrate from the immobilized avidin, streptavidin or their derivativesin a completely unmodified form. Once the biotin is photocleaved from aprotein or protein/binding complex, all the native properties andfunction will be restored to its native form for further use andcharacterization. Choice of photolabile group and/or spacer arm dependson the target growth factor including to which the photocleavable moietyis to be attached. It also depends on the exact conditions forphotocleavage.

Photocleavage of conjugates should typically not damage the releasedtherapeutic agent or impair the agent's activity. Proteins, nucleicacids and other protective groups used in peptide and nucleic acidchemistry are known to be stable to most wavelengths of radiation above300 nm. The yield and exposure time necessary for release of substratephoto-release are strongly dependent on the structure of photoreactivemoiety. For instance, illumination times may vary from about 1 minute toabout 24 hours, less than 4 hours, less than two hours, and less thanone hour, and yields may be between about 1% to about 95%. In someembodiments, the illumination times are from about 10 seconds to about 5minutes, from about 30 seconds to about 5 minutes, from about 1 minuteto about 10 minutes, from about 5 minutes to about 30 minutes, or fromabout 30 minutes to about 90 minutes.

Another embodiment is directed to caged growth factors which arepharmaceutical compositions. Compositions must be safe and nontoxic andcan be administered to patients such as humans and other mammals.Compositions may be mixed with a pharmaceutically acceptable carriersuch as water, oils, lipids, saccharides, polysaccharides, glycerols,collagens and combinations thereof and administered to patients.

In one embodiment, after general administration of the composition tothe patient, the site to be treated is exposed to appropriate radiationreleasing substrate which produces a therapeutic response in a patient.Uncoupling from the bioreactive agent at the point of maximal biologicaleffect is an advantage unavailable using current administration orstabilization procedures. In an analogous fashion, other areas of thepatient's body may be protected from the biological effect of thepharmaceutical agent. Consequently, using these conjugates,site-directed and site-specific delivery of a pharmaceutical agent ispossible.

Polymers and Tissue Scaffolds with Entrained Growth Factors

Tissue scaffolds provide a matrix for cells to guide the process oftissue formation in vivo in three dimensions. Synthetic polymers areattractive scaffold materials as they can be readily produced with awide range of reproducible properties and structures. Polymer tissuescaffolds also provide mechanical support against compressive andtensile forces, thus maintaining the shape and integrity of the scaffoldin the environment in which the tissue is implanted.

The morphology of the tissue scaffold can guide the structure of anengineered tissue, including the size, shape and vascularization of thetissue. The proper design of these tissue scaffolds allows them toexhibit the required range of mechanical and biological functions.Synthetic polymeric materials can be precisely controlled in materialproperties and quality. Moreover, synthetic polymers can be processedwith various techniques and supplied consistently in large quantities.The mechanical and physical properties of synthetic polymers can bereadily adjusted through variation of molecular structures so as tofulfill their functions without the use of either fillers or additives.

In one embodiment, a photolabile group that cleaves (or accelerates thecleavage of) a polymer can be used to release a growth factor that isassociated with or encapsulated within a polymer. This is accomplishedby incorporation of photoactive groups in the polymer backbone, or byinclusion of photoacids in the polymer formulation. One advantage ofthis approach is that derivitization or reformulation of a polymer witha photoactive compound is relatively simpler and cheaper than cagingmultiple growth factors (each demanding a separate synthesis), and suchpolymer can be generalized for use in other systems.

A variety of synthetic biodegradable polymers can be utilized tofabricate tissue scaffolds. In general, these materials are utilized asstructural elements in the scaffold. Poly(glycolic acid) (PGA),poly(lactic acid) (PLA), Poly lactic co-lactic acid (PLLA) andpoly(lactic acid)-poly(glycolic acid) (PLGA) polymers are commonly usedsynthetic polymers in tissue engineering. These polymers are alsoextensively utilized in other biomedical applications such as drugdelivery and are FDA approved for a variety of applications. A number ofPGA, PLA, PLLA and PLGA and other synthetic polymer tissue scaffolds areknown in the art.

The tissue scaffolds may also include natural polymer materials, such ascollagen. Type I collagen may also be combined with glycosaminoglycansto form gels which mimic native dermal tissue. A variety of other ECMmolecules, including laminin, have been utilized as cell delivery tissuescaffolds, and any such tissue scaffold may be used in the context ofthe present disclosure.

In illustrative embodiments, the polymer used in the tissue scaffold isa polymer of lactic acid. Polymers and copolymers of lactic acid aretransparent, colorless thermoplastics with a wide range of physicalproperties that mimic those of many conventional thermoplastics. Whenexposed to moisture or biological fluids, these modified plasticshydrolyze slowly, over a period of several months to natural, harmless,materials such as lactic acid. The copolymers of lactic acid andglycolic acid were originally developed and marketed as an industrialproduct as resorbable sutures. These polymers and copolymers have highstrength and biocompatibility and have controlled degradability.

Poly (lactic acid) and poly (glycolic acid) can be prepared by eithercondensation polymerization of the free acids or by catalytic,ring-opening polymerization of the dilactones. Both polylactic acid andpolyglycolic acid are environmentally compatible because they degraderespectively to lactic acid and glycolic acid, both natural harmlessproducts. While these polymers degrade primarily by hydrolysis, with theaddition of certain other materials, they may degrade also by exposure aother source of light, e.g., UV light. The physical properties such ascrystallinity, melting point, degradation rate, elasticity and the likecan be varied depending upon the amount and the type of copolymerformed.

Incorporation of photoactive compounds into polymer matrix can beaccomplished by the host-guest approach, as well as via chemical bondingof the photoactive compounds to the polymeric backbone. In oneembodiment, the photoactive compound that is incorporated into thepolymer matrix is a keto-containing monomer, which loses CO onirradiation with UV light, in order to fragment the polymer. Forexample, the keto-containing monomer may be a 3-pentanone, such as1,5-bis(4′-methoxycarbonylphenyl)-3-pentanone. In some embodiments, theketone is present in the polymer in an amount of 0.1-100 mole percent,0.1-15 mole percent, or 1.0-5 mole percent of the repeating units. Inanother embodiment, the photosensitive monomer is a methylenebutyrolactone derivative. Further examples of photoactive compounds thatmay be incorporated into polymer matrix are described in U.S. Pat. Nos.3,878,169; 4,883,857; U.S. Pat. No. 5,395,692; and U.S. Pat. No.5,434,236.

Degradation of the polymers may also be induced by a photoacid.Compounds producing acids upon illumination with light are calledphotoacid generators. There are two major groups of acid generators:ionic and non-ionic ones. One suitable group of ionic acid generatorsconsists of onium salts containing metal halides (BF⁻, SbF6⁻, AsF6⁻ orPF₆ ⁻). In one embodiment, the photoacid isaryldiazonium. Uponirradiation with UV light in the range of about 190-300 nm, the oniumsalts photoproduce a protic acid, which may then cleave the polymerbackbone and release the entrained growth factor. In some embodiments,the photoacid generators are selected from the group consisting of:(4-Bromophenyl)diphenylsulfonium triflate;(4-Chlorophenyl)diphenylsulfonium triflate;(4-Fluorophenyl)diphenylsulfonium triflate;(4-Iodophenyl)diphenylsulfonium triflate;(4-Methoxyphenyl)diphenylsulfonium triflate;(4-Methylphenyl)diphenylsulfonium triflate;(4-Phenoxyphenyl)diphenylsulfonium triflate;(4-Phenylthiophenyl)diphenylsulfonium triflate;(4-Methylthiophenyl)methyl phenyl sulfonium triflate;(4-tert-Butylphenyl)diphenylsulfonium triflate;(tert-Butoxycarbonylmethoxynaphthyl)-diphenylsulfonium triflate;2-(4-Methoxystyryl)-4,6-bis(trichloromethyl)-1,3,5-triazine;Bis(4-tert-butylphenyl)iodonium p-toluenesulfonate;Bis(4-tert-butylphenyl)iodonium perfluoro-l-butanesulfonate;Bis(4-tert-butylphenyl)iodonium triflate;Boc-methoxyphenyldiphenylsulfonium triflate; Diphenyliodonium9,10-dimethoxyanthracene-2-sulfonate; Diphenyliodonium nitrate;Diphenyliodonium p-toluenesulfonate; Diphenyliodonium triflate;N-Hydroxy-5-norbornene-2,3-dicarboximide perfluoro-1-butanesulfonate;N-Hydroxynaphthalimide triflate; Triarylsulfonium hexafluorophosphate;Triphenylsulfonium perfluoro-1-butanesufonate; Triphenylsulfoniumtriflate; Tris(4-tert-butylphenyl)sulfonium perfluoro-1-butanesulfonate;and Tris(4-tert-butylphenyl)sulfonium triflate

One aspect of the disclosure incorporates growth factors into the tissuescaffolds or hydrogels to stimulate angiogenesis. It is generallyunderstood that a growth factor that has already been established tohave a beneficial physiological effect on a particular cell type shouldbe chosen for use in regenerating tissue containing such cells. Certaingrowth factors may be used to stimulate the proliferation of a widenumber of cell types, whereas other growth factors may have a morelimited or defined cell-specificity.

Platelet-derived growth factor, (PDGF, e.g., PDGF-BB), is one member ofthe TGF supergene family of growth factors. Particular examples ofsuitable growth factors include other members of the TGF supergenefamily, such as, BMP-2, BMP 4, BMP-7, VEGF, FGF-1, FGF-2, IGF-1, IGF-2,GDF-1, GDF-2, GDF-2, GDF-3, GDF-4, GDF-5, or combinations of the same.In one embodiment, VEGF and PDGF-BB are effective for the growth of newblood vessels and may be used in the present compositions.

The growth factors or stimulatory agents that are useful in the contextof the present disclosure may be purified from natural sources or may berecombinantly prepared proteins. They may be obtained from commercialsources, if desired. Those of skill in the art will know how to obtainand use such growth factors in the context of angiogenesis in light ofthe present disclosure.

Other Agents Added to Polymers and Tissue Scaffolds

One aspect of the disclosure incorporates additional bioactive agentsinto the tissue scaffolds or hydrogels to stimulate tissue growth,relieve pain, fight infection, reduce inflammation or otherwisefacilitate the process of tissue repair and regeneration.

ECM Components. In some embodiments, the tissue-specific function of theproliferating cells that infiltrate the tissue scaffold should bemaintained. The function of the proliferating cells is stronglydependent on the presence of specific growth factors and ECM molecules.For example, in vitro, it is known that cells can be switched from aphase of tissue-specific gene expression to one of proliferation simplyby altering the ECM presentation to the cell. Accordingly ECM proteins,hyaluronic acid or other components of the ECM may be incorporated intothe tissue scaffolds. If desired, any one of a variety of tissuescaffolds that incorporate specific ECM molecules may be used tosupplement the correct signalling to the host's proliferating cells.Synthetic materials that incorporate specific peptides to enhance celladhesion may be used, including those that incorporate a variety ofdifferent peptides in order to mimic the multi-functional nature of ECMmolecules.

Antiinflammatory Agents. In certain embodiments, an anti-inflammatoryagent is combined with the tissue scaffold or hydrogel implanted into asubject. In one embodiment, the tissue scaffold or hydrogel may includethe anti-inflammatory agent alone, while in other embodiments it mayinclude the anti-inflammatory agent in combination with a morphogenicfactor, antibiotic or other biologically active agent. Suitableanti-inflammatory agents include, amongst others, those in the class ofCox-I and Cox II inhibitors. Examples of such agents includeacetyl-salicylic acid, acetaminophens, naproxen, ibuprofen and the like.Another example of a suitable class of anti-inflammatory agents includessoluble cytokine receptors such as EmbrelTM or IL-lb binding receptors.The amount of anti-inflammatory agent used is adjusted so as to bereleased from the tissue scaffold or hydrogel over a period of about 2days or more.

Analgesic Agents or Anesthetics. In some embodiments, the tissuescaffold may include analgesic agents or anesthetics. Theanti-inflammatory agents mentioned above also serve as analgesic agents,thus analgesic agents include anti-inflammatories. In addition, theanalgesic agent may include local pain deadening agents (anesthetics)such as lidocaine, that provide local pain relief for a period of about30 minutes or more.

Antibiotic Agents. In various embodiments, the tissue scaffold mayinclude an antibiotic agent. There are numerous classes of antibioticagents including, but not limited to: tetracyclines, chemically modifiedtetracyclines, cyclosporins, those in the penicillin family, amoxcillan,gentamicin, erythromycin, chloramphenicol, florfenicol, vancomycin,everninomicin, cefotaxime, streptomycin, ciprofloxacin, nalidixic acid,bacitracin, enrofloxacin, and flavomycin.

Photorelease of Caged or Polymer-Entrained Growth Factors

In some embodiments, the process of releasing one or more active growthfactors is done in a spatially selective manner, which allows forcontrolled growth of different sized vascular channels at differentlocations. The timing of release and the rate of release can bemodulated by controlling the intensity (i.e., lumens and duration) ofirradiation. Lastly, the presently disclosed subject matter allows forimproved spatial and temporal control of the release of growth factor.Radiation to promote photorelease of the therapeutic agent can beprovided by a variety of sources including, but not limited to,non-coherent UV light sources and excimer sources.

In an illustrative embodiment, the growth factors VEGF and PDGF areentrained within a tissue engineering scaffold. The scaffold includes atleast two co-polymers, which are synthesized to different photosensitivemonomers, e.g., 5-bis(4′-methoxycarbonylphenyl)-3-pentanone andmethylene butyrolactone. One of the polymers encompasses the VEGF andone of the polymers encompasses the PDGF. The scaffold is implanted in aparticular body tissue and then radiation of the appropriate wavelengthmay be applied to degrade the respective photosensitive monmers.Typically, the polymer containing VEGF will be degraded first, followedby the polymer containing PDGF approximately 2-3 weeks later.

In an illustrative embodiment, about 1-100 fmol VEGF per mm³ of tissueis released per day, over the course of 1 week. In a particularembodiment, about 10 fmol VEGF per mm³ of tissue is released per day,over the course of 1 week. The intensity, duration, and frequency ofadministration of radiation is selected to ensure the desired amount ofVEGF is delivered to the target tissue. The PDGF should be released atapproximately 2-3 weeks, with a release rate of about 10-100fmol/mm³/day. In a particular embodiment about 20 fmol/mm³/day PDGF isreleased. The intensity, duration, and frequency of administration ofradiation is selected to ensure the desired amount of PDGF is deliveredto the target tissue.

The determination of the appropriate dosage of radiation required inorder to degrade the polymer or activate the growth factor may beaccomplished by calibration experiments in which a particular cage orpolymer and growth factor combination is tested in vitro usingdestructive analysis. For example, a particular dosage of radiation isapplied to the tissue scaffold and the sample is analyzed to determinethe amount of cleavage and/or the amount of growth factor released intothe media. The results of the in vitro experiments can be extrapolatedto in vivo conditions.

The release of the growth factors in vivo can also be monitored bytissue biopsy in order to examine the quality of tissue maturation, forexample by looking at the density of vessels (number per squaremillimeter) and their size and size distribution. Non-invasive methodsfor measuring blood vessel density may include angiography orarteriography, and computerized tomography or MRI with radiocontrastdyes (See U.S. Pat. No. 7,011,631).

The radiation applied may comprise one or more wavelengths from theelectromagnetic spectrum including x-rays (about 0.1 nm to about 10.0nm; or about 10¹⁸ to about 10¹⁶ Hz), ultraviolet (UV) rays (about 10.0nm to about 380 nm; or about 8×10¹⁶ to about 10¹⁵ Hz), visible light(about 380 nm to about 750 nm; or about 8×10¹⁴ to about 4×10¹⁴ Hz),infrared light (about 750 nm to about 0.1 cm; or about 4×10¹⁴ to about5×10¹¹ Hz), microwaves (about 0.1 cm to about 100 cm; or about 10⁸ toabout ×10¹¹ Hz), and radio waves (about 100 cm to about 10⁴ m; or about10⁴ to about 10⁸ Hz). Multiple forms of radiation may also be appliedsimultaneously, in combination or coordinated in a step-wise fashion.Radiation exposure may be constant over a period of seconds, minutes orhours, or varied with pulses at predetermined intervals.

Typically, the radiation source is placed at a specified distance fromthe conjugate or polymer to be irradiated. That distance may beempirically determined or calculated from the energy loss producedbetween the source and the target and the amount of energy emitted bythe source. In one embodiment, the radiation applied is UV, visible orIR radiation of the wavelength between about 200 nm to about 1,000 nm,between about 260 nm to about 600 nm, or between about 300 nm to about500 nm. Radiation is administered continuously or as pulses for hours,minutes or seconds, and typically for the shortest amount of timepossible to minimize any risk of damage to the substrate or the patient.Radiation may be administered for less than about one hour, less forthan about 30 minutes, less than about ten minutes, or less than aboutone minute. Visible, UV and IR radiation can be conveniently andinexpensively generated from commercially available sources.

Temporal control may be established by modulating the light intensity onthe construct. Other methods relying on controlled release of the drugswill use polymers of different formulations to obtain different releaserates, and as a result rely on a timing protocol that is set before cellgrowth begins, and does not take account of current conditions in theconstruct. This disclosure enables the release of growth factor inresponse to specific observable biological events, as occurs in nativebiological systems, and, it allows for optimization of releaseconditions using a single formulation, and does not limit the release ofgrowth factors to a smooth rate.

To deliver the growth to a specific body region, a drug delivery devicecan be guided into a position adjacent to the region to be treated,using conventional techniques. After positioning the device adjacent tothe region to be treated, the device can be inflated or expanded so thatits comes into contact with the surrounding tissue. Light is thentransmitted to the target, e.g., by transmission throughout the interiorof the device, causing photolytic release of the therapeutic agent intothe surrounding tissue.

Suitable medical devices include, for example, balloon catheters,endoscopes, polymer stents, and the like. In one embodiment, aconventional balloon angioplasty catheter containing one or more opticalfibers is modified by photoreleasably linking a therapeutic agent to theexterior of the balloon. The catheter is guided into position adjacentto an area to be treated using, for example, a guide wire, and theballoon is then inflated so as to contact and dilate the surroundingtissue. Following inflation of the balloon, radiation from anirradiation source is delivered via one or more optical fibers whichextend through the terminal end of the catheter into the balloon. Adiffusive radio-opaque tip is optionally attached to the terminal endthrough which the radiation is delivered and scattered throughout theballoon. The light delivered through the balloon subsequently causesphotolytic release of one or more growth factors.

Equivalents

While certain embodiments have been illustrated and described, it shouldbe understood that changes and modifications can be made therein inaccordance with ordinary skill in the art without departing from thetechnology in its broader aspects as defined in the following claims.

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and compositions within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can of course vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All references cited herein are incorporated by reference in theirentireties and for all purposes to the same extent as if each individualpublication, patent, or patent application was specifically andindividually incorporated by reference in its entirety for all purposes.

Other embodiments are set forth in the following claims.

1-12. (canceled)
 13. A tissue engineering scaffold comprising: one ormore polymers and at least two growth factors, wherein a first polymeris configured to release a first growth factor in a bioactive state inresponse to a first spectral sensitivity range and a second polymer isconfigured to release a second growth factor in a bioactive state inresponse to a second spectral sensitivity range.
 14. The tissueengineering scaffold of claim 13, wherein the scaffold further comprisescollagen.
 15. The tissue engineering scaffold of claim 13, wherein thescaffold further comprises elastin.
 16. A method of controlling therelease of two or more growth factors at a target site, comprising:delivering the tissue engineering scaffold of claim 13 to a target site;releasing the first growth factor at the target site by radiation of thetarget site with a first wavelength of light in the first spectralsensitivity range; and releasing the second growth factor at the targetsite by radiation of the target site with a second wavelength of lightin the second spectral sensitivity range.
 17. The method of claim 16,wherein an amplitude of the radiation of the target site controls a rateof release of the first growth factor, the second growth factor, orboth.
 18. A method for tissue engineering in a mammal, comprising:applying to a tissue progenitor site of a mammal an effective amount ofa scaffold comprising one or more polymers and at least two growthfactors, wherein a first polymer is configured to release a first growthfactor in a bioactive state in response to a first spectral sensitivityrange and a second polymer is configured to release a second growthfactor in a bioactive state in response to a second spectral sensitivityrange.
 19. The method of claim 18, wherein the scaffold furthercomprises a population of cells.
 20. The method of claim 19, wherein thescaffold comprises a population of vascular endothelial cells or cellprecursors and wherein application of the scaffold to the tissueprogenitor site stimulates vascularization in the site.